Direct selection of recombinant plasmids in Bacillus subtilis.

A system is described which permits the direct, positive selection of recombinant plasmids in Bacillus subtilis. This system relies on the plasmid pBD214 which confers chloramphenicol (Cm) resistance and carries a thy gene, and on BD393, a highly competent B. subtilis thyA thyB host. Thy- strains are resistant to trimethoprim (Tmp), and Thy+ strains are sensitive. Inactivation of the pBD214 thy determinant by insertion of a DNA fragment permits selection of Cmr Tmpr clones, all of which carry recombinant plasmids. This insertional inactivation can be accomplished using the unique EcoRI, BclI, PvuII, or EcoRV sites, all of which are located within the thy gene on pBD214. Some properties of this selective system are described, and its uses for molecular cloning are discussed.